The members of the mechanistically diverse enolase superfamily share a bidomain structure in which a capping domain formed by the N- and C-termini of the polypeptide determines substrate specificity and the functional groups at the C-terminal end of a (beta/alpha)7beta-barrel domain determine reaction mechanism. We want to understand how the structure of the barrel delivers different functions, allowing us to use that information to 1) assist prediction of the functions of unknowns proteins discovered in genome sequencing projects; and2) redesign active sites to catalyze "new" reactions. The project involves four Specific Aims that integrate mechanistic and structural studies. The mechanistic studies will be performed in Dr. Gerlt's laboratory at Illinois (P.I.); the structural studies will be performed in Dr. Rayment's laboratory at Wisconsin (Co-P.I.):1) Structure/function relationships will be established for the newly assigned D-gluconate dehydratases, L-rhamnonate dehydratases, and D-altronate dehydratases in which the active site motifs differ from those previously identified for other dehydratases.2) Functions will be assigned to unknown members by screening purified proteins from several microbial species encoding multiple members for acid sugar dehydratasetisomerase activities.3) Structure/function relationships will be established for o-succinylbenzoate synthases, L-Ala-D/L-Gluepimerases, D-galactonate dehydratases, and D-glucarate dehydratases in which the active site motifs differfrom those previously characterized for "orthologues" that catalyze the same reactions.4) We will test a structural blueprint for functional diversity in the (beta/alpha)7beta-barrel fold by determining whether new functions can be generated by in vitro evolution.